Effective preselection of sex has been accomplished in many species of mammal following the development of safe and reliable methods of separating sperm cells into enriched X chromosome bearing and Y chromosome bearing populations. Separation of X chromosome bearing sperm cells from Y chromosome bearing sperm cells can be accomplished as disclosed herein and as disclosed by various international patent applications, for example: WO 98/34094, WO 99/33956, WO 99/42810, WO 00/06193, WO 01/40765, WO 01/85913, WO 02/43486, WO 02/43574, each hereby incorporated by reference herein.
Now referring to FIGS. 1 and 2, a conventional technology flow cytometer provides a particle or cell source (1) that acts to establish or supply particles or cells (16) (which can be sperm cells or spermatozoa, sperm heads, commonly occurring blood cells such as leukocytes, lymphocytes, monocytes, neutrophils, basophils, macrophages, erythrocytes, platelets, or rare cell types such as fetal cells in circulation within maternal blood, cells which harbor viruses, cancer cells, and the like, as well as parts of cells such as organelles, mitochodria, individual chromosome, as well as man made particles such as beads or microspheres or nanospheres to which a biological component may be bound) that can be stained with at least one fluorochrome for analysis. The particles or cells (16) are introduced within a nozzle (2) in a manner such that the particles or cells (16) are introduced into a fluid stream or sheath fluid (3). The fluid stream (3) is usually supplied by some fluid source (4) so that as the particle or cell source (1) supplies the particles or cells (16) into the fluid (3) they are concurrently fed through the nozzle (2).
In this manner, the fluid stream (3) forms a fluid environment for the particles or cells (16). Since the various fluids are provided to the flow cytometer at some pressure, they exit out of nozzle (2) at a nozzle orifice (5). By providing some type of oscillator (6) which may be very precisely controlled through an oscillator control (7), pressure waves may be established within the nozzle (2) and transmitted to the fluid stream (3) exiting the nozzle (2) at nozzle orifice (5). Since the oscillator (6) acts upon the sheath fluid (3), the fluid stream (8) formed below the nozzle orifice (5) eventually and regularly forms drops (9). Since particles or cells (16) are surrounded by the fluid stream (8) formed below the nozzle orifice, the drops (9) may entrain within them individually isolated particles or cells (16).
Since the drops (9) can entrain individual particles or cells (16), a flow cytometer can be used to separate such particles or cells (16) based upon particle or cell characteristics. This is accomplished through a particle or cell sensing system (10). The particle or cell sensing system involves at least some type of detector or sensor (11) that responds to the particles or cells (16) contained within fluid stream (8). The particle or cell sensing system (10) may cause an action depending upon the relative presence or relative absence of a characteristic, such as fluorochrome bound to the particle or cell or component thereof, such as DNA or lipfds, or mitochondria, or organelles within the cell, that may be excited by an irradiation source such as a laser (12) generating an irradiation beam to which the particle or cell (16) can be responsive.
While each type of particle, cell, or component thereof may be stained with at least one type of fluorochrome, different amounts of fluorochrome(s) bind to each individual particle or cell (16) based on the number of binding sites available to the particular type of fluorochrome used. With respect to spermatozoa, as but one example, the availability of binding sites for Hoechst 33342 stain is dependant upon the amount of DNA contained within each spermatozoa. Because X-chromosome bearing spermatozoa contain more DNA than Y-chromosome bearing spermatozoa, the X-chromosome bearing spermatozoa of a species of mammal can bind a greater amount of fluorochrome than the corresponding Y-chromosome bearing spermatozoa of the same species of mammal. Thus, by measuring the fluorescence emitted by the bound fluorochrome upon excitation, it can be possible to differentiate between X-bearing spermatozoa and Y-bearing spermatozoa.
In order to achieve separation and isolation based upon particle or cell characteristics, emitted light can be received by sensor (11) and fed to some type of separation discrimination system (13) coupled to a droplet charger which differentially charges each droplet (9) based upon the characteristics of the particle or cell (16) contained within that droplet (9). In this manner the separation discrimination system (13) acts to permit the electrostatic deflection plates (14) to deflect drops (9) based on whether or not they contain the appropriate particle or cell (16).
As a result, a flow cytometer acts to separate individual particles or cells (16) entrained in drops (9) by causing them to be directed to one or more collection containers (15). For example, when the separation discrimination system (13) differentiates sperm cells based upon the relative amounts of DNA contained by X-chromosome bearing spermatozoa and Y-chromosome bearing spermatozoa, the droplets entraining X-chromosome bearing spermatozoa can be charged positively and thus deflect in one direction, while the droplets entraining Y-chromosome bearing spermatozoa can be charged negatively and thus deflect the other way, and the waste stream (that is droplets that do not entrain a particle or cell (16) or entrain undesired or unsortable cells) can be left uncharged and thus may be collected in an undeflected stream. Numerous deflection trajectories can be established and collected simultaneously with some conventional flow cytometers.
Even though conventional flow cytometers for the separation of cells or particles have been improved over the past several years significant problems still remain with respect to the resolving capacity of convention flow cytometers.
A significant problem with conventional flow cytometer technology as shown by FIG. 1 may be that the fluid source (4) along with the associated fluid source conduit(s) (23) introduces fluid (3) into the nozzle (2) substantially perpendicular to the flow of the fluid (3) within the nozzle (2). These two directions of flow can interrupt, distort, or delay formation of laminar flow within the nozzle (2). Certain particles or cells (16), being large in comparison to the molecules of the fluid stream, and particularly in cases where the positioning, orientation, and inter-particle distribution is critical to accurate analysis of individual particles or cells (16), and correct entrainment of individual particles or cells (16) into individual drops (9) is required, are strongly influenced by fluid movement which may be turbulent, and therefore maintaining laminar flow is an important aspect, which has been overlooked in the design of conventional flow cytometer technology. It is also noteworthy that the oscillator (6) which serves the primary function of providing pressure waves to allow the formation of individual drops (9), will also provide standing waves of pressure within the nozzle (2) which will influence the laminar flow characteristics, especially as improper nozzle design can lead to harmonic divergences such as pressure beats or sub-oscillations.
A second significant problem with conventional flow cytometer technology as shown by FIG. 1 may be that the cell source conduit (17) between the cell source (1) and the nozzle (2) is not straight. A substantial bend in the cell source conduit (17) can result in a change in fluid pressure in response to the bend in the cell source conduit or can create fluid streams having areal cross sections that exhibit disparate stream velocity. This problem can be exacerbated by cells or debris aggregating at the bend(s) in the cell source conduit (17).
A third significant problem with conventional flow cytometer technology as shown by FIG. 1 may be that the cell source conduit (17) is too long. Conventional flow cytometer cell source conduit (17) from the cell source (1) to the injection point (18) of particles or cells into the fluid stream can be greater than four inches in length. Conventional length cell source conduit (17) can cause cells to settle or aggregate in, or flow through, the cell source conduit in a manner that increases turbulent flow, and therefore decreases apparent resolution of cell populations.
A fourth significant problem with conventional flow cytometer technology as shown by FIG. 1 may be that the cell source conduit (17) and in particular the particle injector (19) portion of the cell source conduit may not be individually replaceable. As such, a failure, or reduced performance, of the cell source conduit (17) or the particle. injector (19) portion can result in the necessity to replace the entire nozzle (2) and the cell source conduit (17) along with any other component of the flow cytometer inseparably joined to the nozzle (2).
A fifth significant problem with conventional flow cytometer technology can be that there may be a connector (20) mounted to the entry end of the particle injector (19) portion of the cell source conduit (17) to couple the injector portion (19) with the cell source fluid conduit (17). Even zero-dead volume connectors or couplers can introduce sufficient deformation or non-concentricity to the cell source conduit interior surface to allow particles or debris to adhere, cling, attach, or otherwise become immobilized at the location of the connector (20) resulting in potential cross contamination between sample populations transferred in the cell source fluid stream, or causing restrictions or otherwise altering the configuration of the cell source fluid path.
A sixth significant problem with convention flow cytometer technology can be that the interior surfaces of the cell source conduit (17) or the interior surfaces of the injector (19) portion of the cell source conduit (17) are sufficiently rough or uneven to reduce apparent resolution of mixed populations of cells or particles. One aspect of this problem can be that surface features that result in the rough or uneven surface can on occasion break away from the cell source conduit interior surface and can become lodged in the flow path. In some cases, restriction or occlusion of the cell source conduit (17) can result. Another aspect of this problem can be that particles interact with the rough or uneven features of the cell source conduit to create asymmetries in the velocity of the fluid stream, which can introduce sheer forces or turbulent flow properties that decrease apparent resolution of cell populations.
A seventh significant problem with conventional flow cytometry technology can be that areal cross sections of the nozzle (2) can be too large. The larger the area of the cross section of the nozzle (2), the greater the area on which bubbles or debris or particles (16) can attach and interfere with the fluid dynamics within the nozzle assembly, and the greater the complexity of standing pressure waves and sub-harmonic pressure waves which may be stabilized in the nozzle (2).
An eight significant problem with conventional flow cytometery may be that the body of the nozzle (2) comprises a first nozzle body element (21) and a second nozzle body element (22). This can create an interior surface of the nozzle having sufficient distortion or roughness to disrupt or diminish the laminar flow of the fluid stream which can translate into reduced resolving capacity of conventional flow cytometer(s), or if the two fabrication materials of a first nozzle body element (21) and a second nozzle body element (22) have different elasticities, they may each deliver pressure waves from the oscillator (6) with different sub-harmonic characteristics, which may tend to distort laminar flow in the critical area of the particle flow path just prior to exiting the nozzle (2) at the orifice (5).
A ninth significant problem in the conventional flow cytometry technology relates to the difficulty in accurate measurement of DNA in particles or cells, such as live mammalian sperm, which bind different amounts of fluorochrome based on known differences in DNA content, and yet have large coefficients of variation which obscure the measurement For example, as shown by Johnson et al., Theriogenology, Vol. 52, No. 8, 1326 (1999) the effect of the size of an X chromosome on total DNA content of a live sperm cell in comparison to the effect of the Y chromosome size on the total DNA of a live sperm cell is determined by the differences between the sizes of an X and a Y chromosome in a specific mammalian species, as well as the actual amount of DNA (numbers and sizes of all other chromosomes). And specifically (X-Y)/X of: 2.8% in humans, 3.0% in rabbits, 3.6% in boars (pig), 3.7% in stallion (horse), 3.8% in bull (cattle), 3.9% in dog, 4.2% in ram (sheep), and as much as 7.5% in chinchilla. Welch et al. show, however, that the coefficient of variation (CV) in the analysis of sperm by conventional methods is considered good vat 0.9% and can even be as high as 1.97%. Welch et al., Theriogenology, Vol. 52, No. 8, 1348 (1999) Thus, especially for species such as humans with very low (X-Y)/X values of 2.8%, it is critical to reduce the CV to lower than 1%, and preferably to as close to zero as possible. The definition of resolution, as used in the description of the instant invention, refers to the capability of the instrument in resolving of sperm into two populations based on the proximity of the measured value from each single sperm to a known value, with the primary negative determinant being a high CV for the population being analyzed.
The instant invention addresses the variety of problems associated with reduced resolution of conventional flow cytometer instruments in separating flow separable particles or cells (16), cells, and specifically sperm cells into enriched populations based upon particle characteristics, or in the context of sperm cells into enriched X-chromosome bearing and Y-chromosome bearing populations.